The present invention relates to methods for capturing a polynucleotide which may be present in a sample onto a solid support. The invention is particularly useful for separating a target polynucleotide from other material in a sample, and is preferably employed as part of a diagnostic procedure that included nucleic acid amplification to detect the presence of the target polynucleotide.
A target polynucleotide is a polynucleotide present in a sample that can be purified from one or more sample components and/or whose presence can be detected using different techniques. Such techniques are typically carried out as part of a diagnostic procedure to detect the presence of a target polynucleotide which is indicative of the presence of an infectious agent or pathogenic condition.
The presence of a target polynucleotide base sequence region, present in a target polynucleotide, can be detected by various methods such as those using nucleic acid probes that hybridize to a target sequence. Probes can be designed to detect different target sequences such as those characteristic of microorganisms, viruses, human genes, plant or animal genes, and/or pathogenic conditions.
A technique for purifying a target polynucleotide, which is often used in diagnostic procedures, involves capturing a target polynucleotide onto a solid support. The solid support retains the target polynucleotide during one or more washing steps of the target polynucleotide purification procedure.
Ranki et al., U.S. Pat. No. 4,486,539 describe a hybridization sandwich technique for capturing and for detecting the presence of a target polynucleotide. The technique involves the capture of the target polynucleotide by a probe bound to a solid support and hybridization of a detection probe to the captured target polynucleotide. Detection probes not hybridized to the target polynucleotide are readily washed away from the solid support. Thus, remaining label is associated with the target polynucleotide initially present in the sample.
Stabinsky, U.S. Pat. No. 4,751,177 describes a method that uses a mediator polynucleotide that hybridizes to both a target polynucleotide and to a polynucleotide fixed on a solid support. The mediator polynucleotide joins the target polynucleotide to the solid support to produce a bound target. A labeled probe can be hybridized to the bound target and unbound labeled probe can be washed away from the solid support.
Englehardt et al., U.S. Pat. Nos. 4,894,324 and 5,288,609, describe a method for detecting a target polynucleotide. The method utilizes two single-stranded polynucleotide segments complementary to the same or opposite strands of the target and results in the formation of a double hybrid with the target polynucleotide. In one embodiment, the hybrid is captured onto a support.
Cape et al., EP Pat. Pub. No. 0 370 694, describe methods and kits for detecting nucleic acids using a solid phase capture means. The methods use oligonucleotide primers labeled with specific binding partners to immobilize primers and primer extension products. The label specifically complexes with its receptor which is bound to a solid support.
According to one aspect of the invention, there is disclosed a method of capturing a target polynucleotide present in a sample. The method includes the steps of incubating a mixture comprising a target polynucleotide, a capture probe, and an immobilized probe in a first hybridization condition that favors formation of a capture probe:target hybridization complex that includes the capture probe and the target polynucleotide, wherein the first hybridization condition disfavors formation of an immobilized probe:capture probe hybridization complex that includes the immobilized probe and the capture probe; and then incubating the mixture in a second hybridization condition that favors formation of the immobilized probe:capture probe hybridization complex, thereby capturing the target polynucleotide in an immobilized probe:capture probe:target hybridization complex that includes the immobilized probe, the capture probe and the target polynucleotide. In one embodiment, the first incubating step uses a temperature below a Tm of the capture probe:target hybridization complex and above a Tm of the immobilization probe:capture probe hybridization complex, and the second incubating step uses a temperature below a Tm of the immobilization probe:capture probe hybridization complex. Preferably, the second incubating step is achieved by lowering the temperature of the first hybridization condition by at least about 10xc2x0 C., or by at least about 20xc2x0 C. In one preferred embodiment the first incubating step uses a temperature of about 60xc2x0 C. and the second incubating step uses a temperature of about 40xc2x0 C. or lower. Alternatively, the first incubating step may use a solution having a chemical stringency that favors formation of the capture probe:target hybridization complex and disfavors formation of the immobilization probe:capture probe hybridization complex, and the second incubating step lowers the chemical stringency of the solution, thereby favoring formation of the immobilization probe:capture probe hybridization complex. One embodiment of the method also includes the step of purifying the immobilized probe:capture probe:target hybridization complex. Another embodiment includes the step of detecting the target polynucleotide in the purified immobilized probe:capture probe:target hybridization complex. Preferably, the detecting step comprises hybridizing a labeled probe to the target polynucleotide in the purified immobilized probe:capture probe:target hybridization complex. In another embodiment, the detecting step further includes removing the labeled probe that has not hybridized to the target polynucleotide. One embodiment of the method also includes the step of detecting the target polynucleotide in the immobilized probe:capture probe:target hybridization complex, preferably by hybridizing a labeled probe to the target polynucleotide. This embodiment may also include the step of removing the labeled probe that has not hybridized to the target polynucleotide. Another embodiment of the method includes the step of amplifying the target polynucleotide to produce an amplified nucleic acid. Preferably, the amplifying step is accomplished by transcription-associated amplification. This embodiment may further include the step of detecting the amplified nucleic acid. Preferably, the detecting step includes hybridizing a labeled probe to the amplified nucleic acid that has a sequence complementary to the target polynucleotide sequence, and may also include removing the labeled probe that has not hybridized to the amplified nucleic acid. In one embodiment of the method the immobilized probe includes a capture probe-binding region of at least five nucleotide base recognition groups in length, and the capture probe includes an immobilized probe-binding region of at least five nucleotide base recognition groups in length, provided that the capture probe-binding region is complementary to the immobilized probe-binding region. Preferably, the capture probe-binding region of the immobilized probe includes (a) a first backbone containing at least one sugar-phosphodiester linkage, or at least one peptide nucleic acid group, at least one phosphorothioate linkage, or a combination thereof, and (b) at least ten nucleotide base recognition groups joined to the first backbone, wherein each nucleotide base recognition group is capable of hydrogen bonding with adenine, guanine, cytosine, thymine, uracil or inosine; and the immobilized probe-binding region of the capture probe includes (a) a second backbone containing at least one sugar-phosphodiester linkage, or at least one peptide nucleic acid group, at least one phosphorothioate linkage, or a combination thereof, and (b) at least ten nucleotide base recognition groups joined to the second backbone, which are capable of hydrogen bonding to the nucleotide base recognition groups joined to the first backbone. In a preferred embodiment, the capture probe-binding region of the immobilized probe consists of a repetitious base sequence of at least 10 nucleotide base recognition groups, and the immobilized probe-binding region of the capture probe consists of a repetitious base sequence comprising at least 25 nucleotide base recognition groups, of which at least 10 nucleotide base recognition groups are complementary to the capture probe-binding region. In one embodiment, the capture probe-binding region includes a sequence of about fourteen contiguous A or T, and the immobilized probe-binding region includes a sequence of 30 bases complementary thereto. In the method, the capture probe and the immobilized probe may each be made up of deoxynucleotides, ribonucleotides, 2xe2x80x2-methoxy substituted nucleotides, 2xe2x80x2-halo substituted nucleotide components, or combinations thereof.
Another aspect of the invention is a method for determining the presence of a target polynucleotide in a sample. This method includes the steps of: providing a capture probe capable of hybridizing to a target polynucleotide present in a sample; mixing the capture probe with a sample suspected of containing the target polynucleotide at a first incubation temperature that favors hybridization of the capture probe and the target polynucleotide, thereby producing a capture probe:target polynucleotide complex; providing an immobilized probe capable of hybridizing to the capture probe; incubating a mixture comprising the capture probe:target polynucleotide complex and the immobilized probe at a second incubation temperature that favors hybridization of the immobilized probe and the capture probe, thereby producing a captured target polynucleotide comprising an immobilized probe:capture probe:target polynucleotide complex; purifying the captured target polynucleotide, thereby producing a purified target polynucleotide; amplifying the purified target polynucleotide, thereby producing an amplified nucleic acid; and detecting the amplified nucleic acid which indicates the presence of the target polynucleotide in the sample. In one embodiment, the detecting step detects an amplified nucleic acid that is complementary to the target polynucleotide or a portion thereof. Preferably, the detecting step comprises using a labeled probe to detect the amplified nucleic acid.
One aspect of the invention is a method for amplifying a target polynucleotide that is present in a sample that includes the steps of:
a. providing a capture probe that hybridizes specifically to a target polynucleotide present in a sample;
b. providing a first primer oligonucleotide that hybridizes specifically to the target polynucleotide at a location different than the location that the capture probe hybridizes to the target polynucleotide;
c. mixing the capture probe and the primer oligonucleotide with a sample suspected of containing the target polynucleotide at a first hybridization condition that favors hybridization of the capture probe to the target polynucleotide and the primer oligonucleotide to the target polynucleotide, thereby producing a capture probe:target polynucleotide:primer oligonucleotide complex formed by hybridization of the capture probe and the primer oligonucleotide to the target polynucleotide, wherein the first hybridization condition disfavors hybridization of an immobilized probe and the capture probe;
d. providing an immobilized probe that hybridizes specifically to the capture probe at a second hybridization condition that is different from the first hybridization condition;
e. incubating a mixture containing the capture probe:target polynucleotide:primer oligonucleotide complex and the immobilized probe at a second hybridization condition that favors hybridization of the immobilized probe and the capture probe, thereby producing a captured target polynucleotide complex consisting essentially of an immobilized probe:capture probe:target polynucleotide:primer oligonucleotide complex formed by hybridization of the immobilized probe to the capture probe in the capture probe:target polynucleotide:primer oligonucleotide complex;
f. purifying the captured target polynucleotide complex from other sample components by washing the captured target polynucleotide complex, thereby producing a purified target polynucleotide retained in the immobilized probe:capture probe:target polynucleotide:primer oligonucleotide complex; and
g. then amplifying a base sequence in the target polynucleotide by initiating nucleic acid polymerization from the first primer oligonucleotide using the target polynucleotide as a template and a DNA polymerase activity, thereby producing an amplified nucleic acid. In one embodiment, the first hybridization condition uses an incubation temperature below a Tm of the capture probe:target polynucleotide hybridization complex and above a Tm of the immobilized probe:capture probe hybridization complex, and the second hybridization condition uses an incubation temperature below a Tm of the immobilized probe:capture probe hybridization complex. Another embodiment includes in the amplifying step hybridizing a second primer oligonucleotide to the target polynucleotide upstream of the first primer oligonucleotide hybridized to the target polynucleotide, and polymerizing a first complementary nucleic acid strand initiating from the first primer oligonucleotide and a second complementary nucleic acid strand initiating from the second primer oligonucleotide, both complementary nucleic acid strands polymerized using the same strand of the target polynucleotide as a template. In one embodiment, the second complementary nucleic acid strand displaces the first complementary nucleic acid strand from the target polynucleotide. In the method, the DNA polymerase activity may be provided by a reverse transcriptase and the first primer oligonucleotide may be a promoter primer. In a preferred embodiment, hybridization of the capture probe to the target polynucleotide and hybridization of the primer oligonucleotide to the target polynucleotide occurs in liquid phase. In one embodiment, the amplifying step initiates polymerization from the first primer oligonucleotide hybridized to the target polynucleotide without releasing the target polynucleotide from the immobilized probe:capture probe:target polynucleotide:primer oligonucleotide complex.
Another aspect of the invention is a method for amplifying a target polynucleotide that is present in a sample, that includes the steps of:
a. providing a capture probe that hybridizes specifically to a target polynucleotide present in a sample;
b. providing a first primer oligonucleotide that hybridizes specifically to the target polynucleotide at a location different than the location that the capture probe hybridizes to the target polynucleotide;
c. mixing the capture probe and the primer oligonucleotide with a sample suspected of containing the target polynucleotide at a first hybridization condition that favors hybridization of the capture probe and the target polynucleotide, thereby producing a capture probe:target polynucleotide:primer oligonucleotide complex formed by hybridization of the capture probe and the primer oligonucleotide to the target polynucleotide, wherein the first hybridization condition disfavors hybridization of an immobilized probe and the capture probe and disfavors hybridization of the primer oligonucleotide to the target polynucleotide;
d. providing an immobilized probe that hybridizes specifically to the capture probe at a second hybridization condition that is different from the first hybridization condition;
e. incubating a mixture containing the capture probe:target polynucleotide complex and the primer oligonucleotide and immobilized probe at a second hybridization condition that favors hybridization of the immobilized probe with the capture probe and the primer oligonucleotide with the target polynucleotide, thereby producing a captured target polynucleotide complex consisting essentially of an immobilized probe:capture probe:target polynucleotide:primer oligonucleotide complex formed by hybridization of the immobilized probe with the capture probe which is hybridized with the target polynucleotide and the primer oligonucleotide with the target polynucleotide which is hybridized to the capture probe;
f. purifying the captured target polynucleotide complex from other sample components by washing the captured target polynucleotide complex, thereby producing a purified target polynucleotide retained in the immobilized probe:capture probe:target polynucleotide:primer oligonucleotide complex; and
g. then amplifying a base sequence in the target polynucleotide by initiating nucleic acid polymerization from the first primer oligonucleotide using the target polynucleotide as a template and a DNA polymerase activity, thereby producing an amplified nucleic acid. In one embodiment of this method, the first hybridization condition uses an incubation temperature below a Tm of the capture probe:target polynucleotide hybridization complex and above a Tm of the immobilized probe:capture probe hybridization complex, and the second hybridization condition uses an incubation temperature below a Tm of the immobilized probe:capture probe hybridization complex. In one embodiment of the method, the amplifying step also hybridizes a second primer oligonucleotide to the target polynucleotide upstream of the first primer oligonucleotide hybridized to the target polynucleotide, and polymerizes a first complementary nucleic acid strand initiating from the first primer oligonucleotide and a second complementary nucleic acid strand initiating from the second primer oligonucleotide, both complementary nucleic acid strands polymerized using the same strand of the target polynucleotide as a template. The second complementary nucleic acid strand can displace the first complementary nucleic acid strand from the target polynucleotide. In a preferred embodiment, the DNA polymerase activity is provided by a reverse transcriptase. In one embodiment, the first primer oligonucleotide is a promoter primer. In another embodiment, the amplifying step initiates polymerization from the first primer oligonucleotide hybridized to the target polynucleotide without releasing the target polynucleotide from the immobilized probe:capture probe:target polynucleotide:primer oligonucleotide complex.
Another aspect of the invention is a method for detecting the presence of a target polynucleotide in a sample, that includes the following steps:
a. providing a capture probe that hybridizes specifically to a target polynucleotide present in a sample;
b. providing a first primer oligonucleotide that hybridizes specifically to the target polynucleotide at a location different than the location that the capture probe hybridizes to the target polynucleotide;
c. mixing the capture probe and the primer oligonucleotide with a sample suspected of containing the target polynucleotide to produce a mixture;
d. incubating the mixture at a first hybridization condition that favors hybridization of the capture probe to the target polynucleotide, thereby producing a capture probe:target polynucleotide complex formed by hybridization of the capture probe and the primer oligonucleotide to the target polynucleotide, wherein the first hybridization condition optionally favors hybridization of the primer oligonucleotide to the target polynucleotide and disfavors hybridization of an immobilized probe and the capture probe;
e. providing an immobilized probe that hybridizes specifically to the capture probe at a second hybridization condition that is different from the first hybridization condition;
f. incubating a mixture containing the capture probe:target polynucleotide complex and the immobilized probe at a second hybridization condition that favors hybridization of the immobilized probe and the capture probe and favors hybridization of the primer oligonucleotide with the target polynucleotide, thereby producing a captured target polynucleotide complex consisting essentially of an immobilized probe:capture probe:target polynucleotide:primer oligonucleotide complex formed by hybridization of the immobilized probe to the capture probe in the capture probe:target polynucleotide complex and the capture probe:target polynucleotide:primer oligonucleotide complex;
g. purifying the captured target polynucleotide complex from other sample components by washing the captured target polynucleotide complex, thereby producing a purified target polynucleotide retained in the immobilized probe:capture probe:target polynucleotide:primer oligonucleotide complex;
h. then amplifying a base sequence in the target polynucleotide by initiating nucleic acid polymerization from the first primer oligonucleotide using the target polynucleotide as a template and a DNA polymerase activity, thereby producing an amplified nucleic acid; and
i. detecting the amplified nucleic acid which indicates the presence of the target polynucleotide in the sample. In one embodiment, the detecting step detects an amplified nucleic acid that is complementary to a sequence in the target polynucleotide. Another embodiment uses a labeled probe to detect the amplified nucleic acid in a homogeneous detection method. In a preferred embodiment, the capture probe is an oligonucleotide containing a target polynucleotide-binding region that is substantially complementary to a sequence in the target polynucleotide, and an immobilized probe-binding region that is substantially complementary to the immobilized probe. One preferred embodiment, in the amplifying step, uses a transcription-associated amplification in which a reverse transcriptase acts together with the first primer oligonucleotide which is a promoter-primer to form a DNA duplex specific for the target polynucleotide and containing a double-stranded promoter, and then an RNA polymerase recognizes the double-stranded promoter to generate multiple RNA transcripts specific for the target polynucleotide, thereby producing the amplified nucleic acid.